The objective of this study was to evaluate the influence of insertion site of the metal binding motif on the bioaccumulation capacity of the hybrid CS3 pili displayed on the surface of Escherichia coli using both computational and experimental methods. Two metal binding motifs (cadmium binding motif (cbm) and cadmium binding beta motif (cb beta m)), identified by searching against the PROSITE database, were inserted into five putative permissive sites of CstH protein (CS3 pili subunit) by using SOEing PCR technique. The expression and surface display of the hybrid pili were evaluated using dot and Western blotting methods and also immunofluorescence microscopy. The cadmium binding affinity and selectivity of the recombinant bacteria displaying various hybrid pili were evaluated using atomic absorption procedure. The results showed that the cadmium binding motifs enabled the cells to sequester cadmium 8- to 16-fold higher than the E. coli expressing native pili. The location of the metal binding motifs in the pili subunit had also a significant effect on the metal-binding properties of the hybrid pili. The insertion at positions 107-108 and 92-93 of the mature CstH showed the highest adsorption in comparison to other positions.